Recently, a large number of measurements using intermolecular interactions such as immune responses are being carried out in clinical tests, etc. However, since conventional methods require complicated operations or labeling substances, several techniques are used that are capable of detecting the change in the binding amount of a test substance with high sensitivity without using such labeling substances. Examples of such a technique may include a surface plasmon resonance (SPR) measurement technique, a quartz crystal microbalance (QCM) measurement technique, and a measurement technique of using functional surfaces ranging from gold colloid particles to ultra-fine particles. The SPR measurement technique is a method of measuring changes in the refractive index near an organic functional film attached to the metal film of a chip by measuring a peak shift in the wavelength of reflected light, or changes in amounts of reflected light in a certain wavelength, so as to detect adsorption and desorption occurring near the surface. The QCM measurement technique is a technique of detecting adsorbed or desorbed mass at the ng level, using a change in frequency of a crystal due to adsorption or desorption of a substance on gold electrodes of a quartz crystal (device). In addition, the ultra-fine particle surface (nm level) of gold is functionalized, and physiologically active substances are immobilized thereon. Thus, a reaction to recognize specificity among physiologically active substances is carried out, thereby detecting a substance associated with a living organism from sedimentation of gold fine particles or sequences.
In all of the above-described techniques, the surface where a physiologically active substance is immobilized is important. Surface plasmon resonance (SPR), which is most commonly used in this technical field, will be described below as an example.
A commonly used measurement chip comprises a transparent substrate (e.g., glass), an evaporated metal film, and a thin film having thereon a functional group capable of immobilizing a physiologically active substance. The measurement chip immobilizes the physiologically active substance on the metal surface via the functional group. A specific binding reaction between the physiological active substance and a test substance is measured, so as to analyze an interaction between biomolecules.
As a thin film having a functional group capable of immobilizing a physiologically active substance, there has been reported a measurement chip where a physiologically active substance is immobilized by using a functional group binding to metal, a linker with a chain length of 10 or more atoms, and a compound having a functional group capable of binding to the physiologically active substance (Japanese Patent No. 2815120). Moreover, a measurement chip comprising a metal film and a plasma-polymerized film formed on the metal film has been reported (Japanese Patent Laid-Open (Kokai) No. 9-264843).
When a physiologically active substance having an amino group is allowed to bind to the surface of a biosensor having carboxylic acid, in general, the carboxylic acid existing on the biosensor surface is activated with 1-(3-dimethylaminopropyl)-3 ethylcarbodiimide (EDC) (which is a water-soluble carbodiimide) and N-hydroxysuccinimide (NHS) in an aqueous medium, and the biosensor surface is then allowed to react with the amino group of the physiologically active substance, so as to form a carboxylic amide. Even when a biosensor surface such as those used in surface plasmon resonance analysis (SPR) or quartz crystal microbalance (QCM) is produced, it has been reported that an amide bond is formed in water by the combination of EDC with NHS (JP Patent Laid-Open (Kokai) Nos. 11-281569 and 2000-39401).
However, when EDC is mixed with NHS in water, there has been a problem that “the stability of the obtained active ester is not sufficient and that it is hydrolyzed over time.”